Global ETD Search

1	Silenciamento gênico por interferência de RNA (RNAi) em traça-do-tomateiro, Tuta absoluta (Meyrick), utilizando bactérias expressando dupla fita de RNA (dsRNA) / Gene silencing by RNA interference (RNAi) in tomato leafminer, Tuta absoluta (Meyrick), using bactéria expressing double-stranded RNA (dsRNA) Bento, Flavia de Moura Manoel 15 December 2017 (has links) O uso da técnica de RNAi vem sendo avaliada em diversos insetos-pragas pois, é uma estratégia inovadora que pode ser integrada no manejo de importantes pragas agrícolas. Os insetos da Ordem Lepidoptera são reconhecidos por apresentarem recalcitrância à técnica de silenciamento utilizando dsRNA. Assim, ajustes devem ser feitos aos métodos de entrega de dsRNA para que haja estabilidade da molécula até atingir o mRNA alvo de silenciamento no inseto. O silenciamento gênico por RNAi possui potencial de uso para o controle da \"traça-do-tomateiro\" Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae), uma das principais pragas do tomateiro no mundo. O objetivo do trabalho foi selecionar e avaliar o silenciamento de genes de T. absoluta, utilizando o método de entrega de dsRNA via bactéria E. coli HT115 (DE3), disponibilizada em dieta artificial. Também, objetivando a aplicabilidade da utilização de bactérias que se desenvolvam no mesmo hábitat de insetos-pragas, estudou-se a colonização das bactérias endofíticas Pantoea agglomerans linhagem 33.1, Burkholderia sp. linhagem SCMS54 e Burkholderia ambifaria linhagem RZ2MS16 em plantas de tomateiro e lagartas de T. absoluta, para posterior transformação e tentativa de utilização como estratégia de entrega de dsRNA para silenciamento de genes alvos de T. absoluta. Foram avaliados, por meio da metodologia de dieta artificial, oito genes de T. absoluta: juvenile hormone inducible protein - JHP; juvenile hormone epoxide hydrolase protein - JHEH; ecdysteroid 25-hydroxylase - PHM; chitin synthase A - CHI; glutathione S-transferase epsilon 2 - GST; carboxylesterase - COE; alkaline phosphatase - AP e; arginine kinase - AK. Por meio de avaliação dos parâmetros biológicos (mortalidade larval; duração da fase larval e peso de pupas) e expressão gênica em cinco períodos de alimentação, comprovou-se a eficiência da metodologia na avaliação do silenciamento gênico por RNAi, sendo possível realizar screening de grande quantidade de genes e avaliar os efeitos do silenciamento gênico no desenvolvimento de T. absoluta. Os genes AK, CHI e JHP apresentaram resultados positivos quanto ao silenciamento gênico e mortalidade larval, sendo promissores para uso de silenciamento por RNAi como estratégia de controle de T. absoluta. Pantoea agglomerans apresentou os melhores resultados de colonização de plantas de tomateiro \"Micro-Tom\" e lagartas de T. absoluta, além de estarem presentes em tecidos preferencialmente utilizados na alimentação das lagartas. Porém, lagartas não apresentaram diferenças na mortalidade larval ao se alimentarem de plantas de tomateiro \"Micro-Tom\" inoculadas com bactérias de P. agglomerans transformadas. / The use of RNAi technique has been evaluated in several insect pests because it is an innovative strategy that can be integrated in the management of important agricultural pests. Insects of the Order Lepidoptera are recognized to present recalcitrance to gene silencing using dsRNA. Thus, adjustments should be done to dsRNA delivery methods to have molecule stability until it reaches the mRNA target for silencing in the insect. Gene silencing by RNAi has potential use to control the tomato leafminer Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae), one of the main insect pests of tomato crop worldwide. The objective of this work was to select and evaluate the silencing of T. absoluta genes using dsRNA delivery method via E. coli HT115 (DE3) bacterium, offered in artificial diet. Also, aiming the applicability of the use of bacteria growing in the same habitat of insect pests, we evaluate the colonization of the endophytic bacteria Pantoea agglomerans strain 33.1, Burkholderia sp. strain SCMS54 and Burkholderia ambifaria strain RZ2MS16 in tomato plants and T. absoluta larvae for further transformation and potential use as dsRNA delivery strategy for silencing target genes of T. absoluta. We evaluated eight genes of T. absoluta: juvenile hormone inducible protein - JHP; juvenile hormone epoxide hydrolase protein - JHEH; ecdysteroid 25-hydroxylase - PHM; chitin synthase A - CHI; glutathione S-transferase epsilon 2 - GST; carboxylesterase - COE; alkaline phosphatase - AP and; arginine kinase - AK. Evaluating biological parameters (larval mortality, larval stage duration and pupal weight) and gene expression in five feeding periods, we proved the efficiency of the methodology in the evaluation of gene silencing by RNAi, and evaluated the effects of gene silencing on the development of T. absoluta. The genes AK, CHI and JHP presented positive results regarding gene silencing and larval mortality, being promising to use RNAi silencing as a strategy to control T. absoluta. Pantoea agglomerans showed good results colonizing \"Micro-Tom\" tomato plants and T. absoluta larvae, besides being present in tissues preferentially used by larvae for feeding. However, larvae did not show differences in larval mortality when feeding on tomato plants inoculated with transformed P. agglomerans. Escherichia coli HT115 (DE3) Escherichia coli HT115 (DE3) Inseto-praga Pest insect Post transciptional silencing RNA de interferência RNA interference Silenciamento pós transcricional
2	Silenciamento gênico por interferência de RNA (RNAi) em traça-do-tomateiro, Tuta absoluta (Meyrick), utilizando bactérias expressando dupla fita de RNA (dsRNA) / Gene silencing by RNA interference (RNAi) in tomato leafminer, Tuta absoluta (Meyrick), using bactéria expressing double-stranded RNA (dsRNA) Flavia de Moura Manoel Bento 15 December 2017 (has links) O uso da técnica de RNAi vem sendo avaliada em diversos insetos-pragas pois, é uma estratégia inovadora que pode ser integrada no manejo de importantes pragas agrícolas. Os insetos da Ordem Lepidoptera são reconhecidos por apresentarem recalcitrância à técnica de silenciamento utilizando dsRNA. Assim, ajustes devem ser feitos aos métodos de entrega de dsRNA para que haja estabilidade da molécula até atingir o mRNA alvo de silenciamento no inseto. O silenciamento gênico por RNAi possui potencial de uso para o controle da \"traça-do-tomateiro\" Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae), uma das principais pragas do tomateiro no mundo. O objetivo do trabalho foi selecionar e avaliar o silenciamento de genes de T. absoluta, utilizando o método de entrega de dsRNA via bactéria E. coli HT115 (DE3), disponibilizada em dieta artificial. Também, objetivando a aplicabilidade da utilização de bactérias que se desenvolvam no mesmo hábitat de insetos-pragas, estudou-se a colonização das bactérias endofíticas Pantoea agglomerans linhagem 33.1, Burkholderia sp. linhagem SCMS54 e Burkholderia ambifaria linhagem RZ2MS16 em plantas de tomateiro e lagartas de T. absoluta, para posterior transformação e tentativa de utilização como estratégia de entrega de dsRNA para silenciamento de genes alvos de T. absoluta. Foram avaliados, por meio da metodologia de dieta artificial, oito genes de T. absoluta: juvenile hormone inducible protein - JHP; juvenile hormone epoxide hydrolase protein - JHEH; ecdysteroid 25-hydroxylase - PHM; chitin synthase A - CHI; glutathione S-transferase epsilon 2 - GST; carboxylesterase - COE; alkaline phosphatase - AP e; arginine kinase - AK. Por meio de avaliação dos parâmetros biológicos (mortalidade larval; duração da fase larval e peso de pupas) e expressão gênica em cinco períodos de alimentação, comprovou-se a eficiência da metodologia na avaliação do silenciamento gênico por RNAi, sendo possível realizar screening de grande quantidade de genes e avaliar os efeitos do silenciamento gênico no desenvolvimento de T. absoluta. Os genes AK, CHI e JHP apresentaram resultados positivos quanto ao silenciamento gênico e mortalidade larval, sendo promissores para uso de silenciamento por RNAi como estratégia de controle de T. absoluta. Pantoea agglomerans apresentou os melhores resultados de colonização de plantas de tomateiro \"Micro-Tom\" e lagartas de T. absoluta, além de estarem presentes em tecidos preferencialmente utilizados na alimentação das lagartas. Porém, lagartas não apresentaram diferenças na mortalidade larval ao se alimentarem de plantas de tomateiro \"Micro-Tom\" inoculadas com bactérias de P. agglomerans transformadas. / The use of RNAi technique has been evaluated in several insect pests because it is an innovative strategy that can be integrated in the management of important agricultural pests. Insects of the Order Lepidoptera are recognized to present recalcitrance to gene silencing using dsRNA. Thus, adjustments should be done to dsRNA delivery methods to have molecule stability until it reaches the mRNA target for silencing in the insect. Gene silencing by RNAi has potential use to control the tomato leafminer Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae), one of the main insect pests of tomato crop worldwide. The objective of this work was to select and evaluate the silencing of T. absoluta genes using dsRNA delivery method via E. coli HT115 (DE3) bacterium, offered in artificial diet. Also, aiming the applicability of the use of bacteria growing in the same habitat of insect pests, we evaluate the colonization of the endophytic bacteria Pantoea agglomerans strain 33.1, Burkholderia sp. strain SCMS54 and Burkholderia ambifaria strain RZ2MS16 in tomato plants and T. absoluta larvae for further transformation and potential use as dsRNA delivery strategy for silencing target genes of T. absoluta. We evaluated eight genes of T. absoluta: juvenile hormone inducible protein - JHP; juvenile hormone epoxide hydrolase protein - JHEH; ecdysteroid 25-hydroxylase - PHM; chitin synthase A - CHI; glutathione S-transferase epsilon 2 - GST; carboxylesterase - COE; alkaline phosphatase - AP and; arginine kinase - AK. Evaluating biological parameters (larval mortality, larval stage duration and pupal weight) and gene expression in five feeding periods, we proved the efficiency of the methodology in the evaluation of gene silencing by RNAi, and evaluated the effects of gene silencing on the development of T. absoluta. The genes AK, CHI and JHP presented positive results regarding gene silencing and larval mortality, being promising to use RNAi silencing as a strategy to control T. absoluta. Pantoea agglomerans showed good results colonizing \"Micro-Tom\" tomato plants and T. absoluta larvae, besides being present in tissues preferentially used by larvae for feeding. However, larvae did not show differences in larval mortality when feeding on tomato plants inoculated with transformed P. agglomerans. Escherichia coli HT115 (DE3) Inseto-praga RNA de interferência Silenciamento pós transcricional Escherichia coli HT115 (DE3) Pest insect Post transciptional silencing RNA interference
3	From protein production to genome evolution in Escherichia coli Schlegel, Susan January 2013 (has links) The aim of my Ph.D. studies was to improve production yields of membrane- and secretory proteins in the widely used E. coli protein production strain BL21(DE3). In this strain expression of the gene encoding the protein of interest is driven by the powerful T7 RNA polymerase (T7 RNAP) whose gene is located on the chromosome and under control of the strong, IPTG-inducible lacUV5 promoter. Unfortunately, the production of many membrane and secretory proteins is 'toxic' to BL21(DE3), resulting in poor growth and low production yields. To understand this ‘toxicity’, the BL21(DE3) derived mutant strains C41(DE3) and C43(DE3) were characterized. Somehow, these strains can efficiently produce many ‘toxic’ membrane and secretory proteins. We showed that mutations weakening the lacUV5 promoter are responsible for this. These mutations result in a slower onset of protein production upon the addition of IPTG, which avoids saturating the Sec-translocon capacity. The Sec-translocon is a protein-conducting channel in the cytoplasmic membrane mediating the biogenesis of membrane proteins and translocation of secretory proteins. Next, we constructed a BL21(DE3)-derivative, Lemo21(DE3), in which the activity of T7 RNAP can be precisely controlled by titrating in its natural inhibitor T7 lysozyme using the rhamnose promoter system. In Lemo21(DE3), the expression level of genes encoding membrane and secretory proteins can be set such that the Sec-translocon capacity is not saturated. This is key to optimizing membrane and secretory protein production yields. Finally, reconstructing the evolution of C41(DE3) from BL21(DE3) in real time showed that during its isolation C41(DE3) had acquired mutations critical for surviving the starvation conditions used, and provided insight in how the mutations in the lacUV5 promoter had occurred. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p> Escherichia coli BL21(DE3) protein production membrane proteins secretory proteins genome evolution
4	Biochemical Study of Engineered Fluorescent Proteins as Calcium Sensors and the Effect of Calcium and PH in Cell Reproduction and Protein Expression Delgado, Malcom Arturo 01 December 2009 (has links) Calcium plays important roles in both eukaryotic and prokaryotic cells. Its actions help to stabilize cell synthesis, growth and development. In this thesis, studies have been completed to determine effects of calcium and pH on bacterial cell growth and protein expression using the bacterial cell strain E.coli BL21(DE3). Our studies demonstrated the addition of calcium addition in the media does not affect growth but increases protein expression, while reducing the pH from 7 to 4 through the addition of 10mM EGTA in LB media inhibits both. Additionally, we report studies on the design, expression, and purification of fluorescent mCherry variants and their differences in their optical properties, including: extinction coefficients , quantum yields and pKa values. Also, we report progress in the crystallization of two GFP calcium sensors: G1 and D1, using 13 and15% PEG 4000 and 3350 respectively in 50mM HEPES buffer (pH 6.8-7.0) in an effort to optimize crystallization. Calcium Protein mCherry Green Fluorescent Protein (GFP) E. coli. X-ray crystallography Protein expression Fluorescent proteins BL21(DE3).
5	Process development for the production of a therapeutic Affibody® Molecule / Processutveckling för att tillverka en Affibody®-molekyl avsedd för cancerterapi Fridman, Belinda January 2014 (has links) Recently HER3, member of the epidermal growth factor receptor family (EGFR), has been found to play a crucial role in the development of resistance towards inhibitors that are given to patients with HER1- and HER2-driven cancers. As HER3 is up-regulated or over-activated in several types of human cancers, it is of outmost importance that new innovative drugs target its oncologic activity. The Affibody® Molecule Z08698 inhibits the heregulin induced signalling of HER3 with high affinity (KD~50 pM). As the Affibody® Molecule is small, has high solubility and outstanding folding kinetics, an effective penetration of tumour tissue is suggested together with a rationalized manufacturing process. Further coupling to an albumin binding domain (ABD) expands the plasma half-life of the molecule, hence increasing the molecule's potential of serving as a therapeutic. A process development for production of Z08698-VDGS-ABD094 has been established, where the molecule is efficiently produced in the E. coli host strain BL21(DE3), through a T7 based expression system. Cultivations were performed with a fed-batch fermentation process and the conditions were further optimized in order to obtain highest expression, while avoiding undesirable modifications like gluconoylations. By employing Design of experiments in combination with multivariate data analysis, a production process resulting in ~3.5 g product/ l culture could be verified. Moreover, thermolysis was evaluated as a suitable method for cell disruption, enabling an easy and cost-effective manufacturing process of the ABD fused Affibody® Molecule. Affibody® Molecule HER3 EGFR HER2 cancer therapy process development DOE multivariate data analysis E. coli ABD plasma half-life thermolysis fed-batch gluconoylation BL21(DE3) T7 expression system
6	Stabil och antibiotikafri läkemedelsproduktion i rekombinant Escherichia coli Benevides, Kristina, Broström, Oscar, Elison Kalman, Grim, Swenson, Hugo, Vlassov, Andrei, Ågren, Josefin January 2017 (has links) Den här rapporten presenterar ett antibiotikafritt, stabilt och kromosombaserat expressionssystem för läkemedelsproduktion i Escherichia coli på beställning av företaget Affibody AB. E. coli-stammen BL21(DE3) valdes som värdorganism för expressionssystemet. Systemet består av en genkassett som innehåller en T7-promotor, en 5′-UTR från genen ompA och en terminatorsekvens från RNA-operonet rrnB. Fyra kopior av genkassetten ska integreras i pseudogenerna caiB, yjjM, hsdS och yjiV. En datormodell som modellerar det egentliga kopietalet i cellerna har skapats i mjukvaran MATLAB, vilket visar att det uppskattas vara maximalt 32 kopior av genkassetten per cell på grund av replikation av kromosomen. Ett högt pH i fermentorn; att använda fed-batch och blandade kolhydratkällor; och att använda stammen BL21(DE3) minskar acetatproduktionen i cellen. En lägre acetatproduktion kan leda till en högre produkthalt. En proteinutbytesmodell för mjukvaran MATLAB har konstruerats för att uppskatta koncentrationen av Affibody®-molekylen i en E. coli cell. copynumber copy-number copy number origin of replication rekombinant proteinexpression läkemedel E. coli antibiotikafri antibiotika expressionssystem expression system escherichia coli kromosom kromosombaserad kromosomal plasmidfri plasmid T7-promotor T7 promotor ompA rrnB autoinduktion auto induktion matlab kopietalmodell utbytemodell stabil pH pseudogen pseudo gen caiB yjjM hsdS yjiV fermentor batch fed-batch kontinuerlig fed batch fermentation Affibody affibody-molekyl affibody-molekyler affibodymolekyl affibodymolekyler bioreaktor acetat acetatbildning acetatproduktion acetatreduktion acetatreducering peptidläkemedel peptider peptid T7-system operator lac-operator operon 5'-UTR 3'-UTR sekundärstruktur mRNA T1 T2 terminator termineringssekvens optimering lac-operon repressor inducering BL21(DE3) BL21 B-stam B stam modell kopietal kopie tal OriC Pharmaceutical Biotechnology Läkemedelsbioteknik

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